Detection of Bordetella

ABSTRACT

The invention provides methods to detect  Bordetella pertussis  and/or  Bordetella parapertussis  in a biological sample. Primers and probes for the differential detection of  B. pertussis  and  B. parapertussis  are provided by the invention. Articles of manufacture containing such primers and probes for detecting  B. pertussis  and/or  B. parapertussis  are further provided by the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part and claims priorityunder 35 U.S.C. §120 of U.S. application Ser. No. 10/062,875 filed Jan.31, 2002, which claims priority under 35 U.S.C. §19(e) of U.S.Application No. 60/265,534 filed Jan. 31, 2001.

TECHNICAL FIELD

[0002] This invention relates to bacterial diagnostics, and moreparticularly to detection of Bordetella.

BACKGROUND

[0003] Whooping cough, caused by Bordetella pertussis, is presently oneof the ten most common causes of death from infectious diseaseworldwide. Patients first present with a common cold and a cough.However, the disease progresses to paroxysmal coughing followed by acharacteristic inspiratory whoop. Secondary symptoms arising frombacterial pneumonia, neurological complications (i.e., seizures andencephalopathy), and pressure effect complications (i.e., pneumothorax,epistaxis, subdural hematomas, hernias, and rectal prolapse) can alsooccur. From the onset of initial symptoms, the disease can take 6-8weeks to resolve. Bordetella parapertussis is closely related to B.pertussis and may cause a similar illness, especially in children;however, the symptoms are less severe and are generally of shorterduration than B. pertussis.

[0004]Pertussis and its associated complications were a major cause ofinfant and childhood mortality until the introduction of thediphtheria-tetanus-pertussis (DTP) vaccine in the 1940s. Widespread useof the vaccine in the American population resulted in a 98% decrease inthe incidence of pertussis. According to the Centers for Disease Control(CDC; Atlanta, Ga.), there has been a resurgence of pertussis, and theincidence of pertussis in the general population has been on the risesince 1991. There was an 82% increase in total cases reported to the CDCin 1993 compared to the same period in 1992 (1992=3004; 1993=5457). In1992, there were outbreaks of pertussis in Massachusetts and Maryland,and in 1994 there was an outbreak of erythromycin-resistant B. pertussisdescribed in Arizona. This trend is also being seen outside the UnitedStates. In 1996, the Netherlands had an outbreak of pertussis, reporting12 times the number of cases seen in 1995 (1995=341; 1996=4231).

[0005] Many cases of B. pertussis go undiagnosed and unreported. Whilepertussis is highly communicable and can cause severe disease, symptomsin older children and adults, including those previously immunized, maybe difficult to differentiate from the nonspecific symptoms ofbronchitis and upper respiratory tract infections. Clinical diagnosis ofpertussis is complicated by the fact that the characteristic cough(whoop) is rarely observed in infants and adult patients.

SUMMARY

[0006] Methods of the invention can be used to rapidly identify B.pertussis and/or B. parapertussis from a biological sample fordifferential diagnosis of pertussis infection. Nasopharyngeal swabs andaspirates can be treated to release the DNA from Bordetella species inthe sample. Using specific primers and probes, the method includesamplifying and monitoring the development of specific template nucleicacid using fluorescence resonance emission technology (FRET).

[0007] In one aspect, the invention provides a method for detecting thepresence or absence of Bordetella pertussis and/or B. parapertussis in abiological sample from an individual. The method includes performing atleast one cycling step of amplifying and hybridizing. The amplifyingstep includes contacting the sample with a pair of IS481 primers and/ora pair of IS1001 primers to produce an IS481 and/or an IS1001amplification product, respectively, if IS481 and/or IS1001 nucleic acidmolecules are present in the sample. The hybridizing step includescontacting the sample with a pair of IS481 probes and/or a pair ofIS1001 probes. Generally, the members within each pair of IS481 andIS1001 probes hybridize within no more than five nucleotides of eachother. Typically, a first IS481 probe of the pair of IS481 probes islabeled with a donor fluorescent moiety and a second IS481 probe of thepair of IS481 probes is labeled with a corresponding acceptorfluorescent moiety. Likewise, a first IS1001 probe of the pair of IS1001probes is labeled with a donor fluorescent moiety and a second IS1001probe of the pair of IS1001 probes is labeled with a correspondingacceptor fluorescent moiety. The donor fluorescent moiety and/or theacceptor fluorescent moieties on the IS481 and the IS1001 probes can bedifferent.

[0008] The method further includes detecting the presence or absence ofFRET between the donor fluorescent moiety of the first IS481 probe andthe corresponding acceptor fluorescent moiety of the second IS481 probeand/or between the donor fluorescent moiety of the first IS1001 probeand the corresponding acceptor fluorescent moiety of the second IS1001probe. The presence of FRET usually indicates the presence of B.pertussis and/or B. parapertussis in the biological sample, while theabsence of FRET usually indicates the absence of B. pertussis or B.parapertussis in the biological sample.

[0009] The method can additionally include determining the meltingtemperature between the IS481 probes and the IS481 amplification productand/or between the IS1001 probes and the IS1001 amplification product.The melting temperature(s) further confirms the presence or absence ofB. pertussis and the presence or absence of B. parapertussis in thesample.

[0010] In another aspect of the invention, the above-described methodcan be performed to detect B. pertussis using primers and probes thathybridize to IS481 nucleic acid molecules. Alternatively, theabove-described method can be performed to detect B. parapertussis usingprimers and probes that hybridize to IS1001 nucleic acid molecules.

[0011] In one aspect of the invention, there is provided a pair of IS481primers including a first IS481 primer and a second IS481 primer. Afirst IS481 primer can include the sequence 5′-CCA GTT CCT CAA GGACGC-3′ (SEQ ID NO:1), and the second IS481 primer can include thesequence 5′-GAG TTC TGG TAG GTG TGA GCG TA-3′ (SEQ ID NO:2). A firstIS481 probe can include the sequence 5′-CAC CGC TTT ACC CGA CCT TAC CGCCCA C-3′ (SEQ ID NO:3), and a second IS481 probe can include thesequence 5′-GAC CAA TGG CAA GGC CGA ACG CTT CAT C-3′ (SEQ ID NO:4). Inanother embodiment, a second IS481 probe can include the sequence 5′-GACCAA TGG CAA GGC TCG AAC GCT TCA TC-3′ (SEQ ID NO:11).

[0012] In another aspect of the invention, there is provided a pair ofIS1001 primers including a first IS1001 primer and a second IS1001primer. A first IS1001 primer can include the sequence 5′-GGC GAT ATCAAC GGG TGA-3′ (SEQ ID NO:5), and the second IS1001 primer can includethe sequence 5′-CAG GGC AAA CTC GTC CAT C-3′ (SEQ ID NO:6). Theinvention further provides a first IS1001 probe that can include thesequence 5′-GTT CTT CGA ACT GGG TTG GCA TAC-3′ (SEQ ID NO:7), and asecond IS1001 probe that can include the sequence 5′-GTC AAG ACG CTG GACAAG GCT C-3′ (SEQ ID NO:8). In another embodiment, a first IS1001 probecan include the sequence 5′-GGT TGG CAT ACC GTC AAG A-3′ (SEQ ID NO:12),and a second IS1001 probe can include the sequence 5′-GCT GGA CAA GGCTCG-3′ (SEQ ID NO:13).

[0013] Representative biological samples include nasopharyngeal swabs,nasopharyngeal aspirates, and throat swabs. Generally, the members ofthe pair of IS481 probes hybridize within no more than two nucleotidesof each other, or within no more than one nucleotide of each other. Arepresentative donor fluorescent moiety is fluorescein, andcorresponding acceptor fluorescent moieties include LC™-Red 640, LC™-Red705, Cy5, and Cy5.5. Additional corresponding donor and acceptorfluorescent moieties are known in the art.

[0014] In one aspect, the detecting step includes exciting thebiological sample at a wavelength absorbed by the donor fluorescentmoiety and visualizing and/or measuring the wavelength emitted by theacceptor fluorescent moiety. In another aspect, the detecting stepincludes quantitating FRET. In yet another aspect, the detecting step isperformed after each cycling step (e.g., in real-time).

[0015] The above-described methods can further include preventingamplification of a contaminant nucleic acid. Preventing amplificationcan include performing amplifying steps in the presence of uracil andtreating the biological samples with uracil-DNA glycosylase prior toamplifying. In addition, the cycling step can be performed on a controlsample. A control sample can include the same portion of the IS481 orIS1001 nucleic acid molecule. Alternatively, a control sample caninclude a nucleic acid molecule other than an IS481 or IS1001 nucleicacid. Cycling steps can be performed on such a control sample using apair of control primers and a pair of control probes that are other thanIS481 or IS1001 primers and probes. One or more amplifying stepsproduces a control amplification product. Each of the control probeshybridize to the control amplification product.

[0016] In yet another aspect, the invention provides articles ofmanufacture, or kits. Kits of the invention can include a pair of IS481primers, a pair of IS481 probes, and a donor and corresponding acceptorfluorescent moiety. For example, a first IS481 primer provided in a kitof the invention can include the sequence 5′-CCA GTT CCT CAA GGA CGC-3′(SEQ ID NO:1), and a second IS481 primer can include the sequence 5′-GAGTTC TGG TAG GTG TGA GCG TA-3′ (SEQ ID NO:2). A first IS481 probeprovided in a kit of the invention can include the sequence 5′-CAC CGCTTT ACC CGA CCT TAC CGC CCA C-3′ (SEQ ID NO:3), and a second IS481 probecan include the sequence 5′-GAC CAA TGG CAA GGC CGA ACG CTT CAT C-3′(SEQ ID NO:4). In another embodiment, a second IS481 probe provided in akit of the invention can include the sequence 5′-GAC CAA TGG CAA GGC TCGAAC GCT TCA TC-3′ (SEQ ID NO:11).

[0017] In another aspect of the invention, there is provided an articleof manufacture, or kit. Kits of the invention can include a pair ofIS481 primers, a pair of IS481 probes, and a donor and correspondingacceptor fluorescent moiety. For example, a first IS1001 primer providedin a kit of the invention can include the sequence 5′-GGC GAT ATC AACGGG TGA-3′ (SEQ ID NO:5), and a second IS1001 primer can include thesequence 5′-CAG GGC AAA CTC GTC CAT C-3′ (SEQ ID NO:6). A first IS1001probe provided in a kit of the invention can include the sequence 5′-GTTCTT CGA ACT GGG TTG GCA TAC-3′ (SEQ ID NO:7), and the second IS1001probe can include the sequence 5′-GTC AAG ACG CTG GAC AAG GCT C-3′ (SEQID NO:8). In another embodiment, a first IS1001 probe provided in a kitof the invention can include the sequence 5′-GGT TGG CAT ACC GTC AAGA-3′ (SEQ ID NO: 12), and a second IS1001 probe provided in a kit of theinvention can include the sequence 5′-GCT GGA CAA GGC TCG-3′ (SEQ IDNO:13).

[0018] Articles of manufacture can include fluorophoric moieties forlabeling the probes or probes already labeled with donor andcorresponding acceptor fluorescent moieties. The article of manufacturecan also include a package insert having instructions thereon for usingthe primers, probes, and fluorophoric moieties to detect the presence orabsence of Bordetella in a biological sample and can further includeinstructions thereon for using the probes to distinguish between B.pertussis and/or B. parapertussis in a biological sample.

[0019] In yet another aspect of the invention, there is provided amethod for detecting the presence or absence of B. pertussis in abiological sample from an individual. Such a method includes performingat least one cycling step. A cycling step can include an amplifying stepand a hybridizing step. Generally, an amplifying step includescontacting the sample with a pair of IS481 primers to produce an IS481amplification product if a B. pertussis IS481 nucleic acid molecule ispresent in the sample. Generally, a hybridizing step includes contactingthe sample with an IS481 probe. Such an IS481 probe is usually labeledwith a donor fluorescent moiety and a corresponding acceptor fluorescentmoiety. The methods further include detecting the presence or absence offluorescence resonance energy transfer (FRET) between the donorfluorescent moiety and the acceptor fluorescent moiety of the IS481probe. The presence or absence of FRET is indicative of the presence orabsence of B. pertussis in said sample. In addition to the IS481 primersand probe described herein, this method also can be performed usingIS1001 primers and probe.

[0020] In one aspect, amplification can employ a polymerase enzymehaving 5′ to 3′ exonuclease activity. Thus, the donor and acceptorfluorescent moieties would be within no more than 5 nucleotides of eachother along the length of the probe. In another aspect, the IS481 probeincludes a nucleic acid sequence that permits secondary structureformation. Such secondary structure formation generally results inspatial proximity between the donor and corresponding acceptorfluorescent moiety. According to this method, the acceptor fluorescentmoiety on a probe can be a quencher.

[0021] In another aspect of the invention, there is provided a methodfor detecting the presence or absence of B. pertussis in a biologicalsample from an individual. Such a method includes performing at leastone cycling step. A cycling step can include an amplifying step and adye-binding step. An amplifying step generally includes contacting thesample with a pair of IS481 primers to produce an IS481 amplificationproduct if a B. pertussis IS481 nucleic acid molecule is present in thesample. A dye-binding step generally includes contacting the IS481amplification product with a nucleic acid binding dye. The methodfurther includes detecting the presence or absence of binding of thenucleic acid binding dye to the amplification product. According to theinvention, the presence of binding is typically indicative of thepresence of B. pertussis in the sample, and the absence of binding istypically indicative of the absence of B. pertussis in the sample. Sucha method can further include the steps of determining the meltingtemperature between the IS481 amplification product and the nucleic acidbinding dye. Generally, the melting temperature confirms the presence orabsence of B. pertussis. Representative double-stranded DNA binding dyesinclude SYBRGreenI®, SYBRGold®, and ethidium bromide.

[0022] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

[0023] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe drawings and detailed description, and from the claims.

DETAILED DESCRIPTION

[0024]B. pertussis, the bacterium causing pertussis or “whooping cough”has traditionally been difficult to detect in a clinically usefulmanner. Several diagnostic methods are available, but most lacksensitivity, require extended culture incubation times for results,and/or require repeated sampling and testing to verify significantincreases of immunoglobulin G antibodies against pertussis toxin orimmunoglobulin A antibodies against B. pertussis in paired sera. Thepresent invention provides methods of detecting B. pertussis and/or B.parapertussis in a biological sample from an individual suspected ofhaving pertussis. The methods feature the ability to distinguish betweenB. pertussis and B. parapertussis. The invention further provides kitscontaining primers and probes to carry out the differential diagnosticmethods of the invention.

[0025]Pertussis

[0026]B. pertussis is transmitted by respiratory droplets and causesdisease only in humans. Virulence factors of B. pertussis includeagglutinogens, fimbriae, P.69/pertactin, pertussis toxin, filamentoushaemagglutinin, adenylate cyclase, tracheal cytotoxin, dermonecrotictoxin, lipopolysaccharide, tracheal colonization factor, serumresistance factor, and type III secretion. Virulence factor expressionis regulated by the bvgAS locus, a two-component signal transductionsystem. The pathophysiological sequence consists of attachment(fimbriae, P.69/pertactin, tracheal colonization factor, pertussistoxin, filamentous haemagglutinin), evasion of host defense (adenylatecyclase, pertussis toxin, serum resistance factor), local effects(tracheal cytotoxin), and systemic effects (pertussis toxin).

[0027] Various methods to diagnose pertussis are available, includingculture, serological methods, and the polymerase chain reaction (PCR).Serotyping of isolates to detect agglutinogens 2 and 3 is useful becauseserotype 1,2 may be associated with higher mortality, and antibodies tothe agglutinins may be protective in both animals and humans. A cellularvaccines containing one to five components are increasingly being usedin various countries. Immunization using whole-cell vaccine is alsoeffective but is reactogenic. Protective immunity to pertussiscorrelates with high levels of antibody to each of pertactin, fimbriae,and pertussis toxin.

[0028]Pertussis is a communicable disease that can be very severe inyoung infants. Early diagnosis and treatment are essential to limit theseverity of the disease and minimize transmission. The wide prevalenceof pertussis and its changing epidemiology has highlighted the need formore sensitive and rapid methods for diagnostic testing. Currentdiagnostic tests for B. pertussis and B. parapertussis are difficult toperform due to the fastidious nature of Bordetella organisms, lacksensitivity, and require 3-5 days of growth to allow identification.Serologic testing by enzyme-linked immunosorbent assay (ELISA) orWestern blot is sensitive and specific, but requires the comparison of 2serum specimens from the subject collected over a 4-week interval.Direct fluorescent antibody testing (DFA) of nasopharyngeal secretionslacks sensitivity. The reference method is direct culture of theorganism from nasopharyngeal secretions, but direct culture ofBordetella has a turnaround time of 1 to 2 days. Further, the organismis susceptible to environmental exposure (changes in temperature anddrying) and has specific growth requirements, making recovery by culturedifficult.

[0029]B. pertussis and B. parapertussis Nucleic Acids andOligonucleotides

[0030] In one embodiment, methods of the invention use the insertionsequence IS481 (GenBank Accession No. M28220; SEQ ID NO:9) to detect B.pertussis in a biological sample. B. pertussis typically contains 50-100copies of IS481. The IS481 sequence was described by McPheat et al. (J.Gen. Microbiol., 135:1515-1520, 1989). In another embodiment, methods ofthe invention use the insertion sequence IS1001 (GenBank Accession No.X66858; SEQ ID NO:10) to detect B. parapertussis in a biological sample.B. parapertussis typically contains 30-35 copies of IS1001. The IS1001sequence was described by van der Zee et al. (J. Bacteriol.,175:141-147, 1993). Bordetella nucleic acids other than thoseexemplified herein (e.g., other than IS481 or IS1001 nucleic acids) alsocan be used to detect Bordetella in a sample and are known to those ofskill in the art. Specifically, primers and probes to amplify and detectB. pertussis IS481 nucleic acid are provided by the invention, as areprimers and probes to amplify and detect B. parapertussis IS1001 nucleicacid.

[0031] Primers that amplify a Bordetella nucleic acid molecule (e.g.,IS481 or IS1001) can be designed using, for example, a computer programsuch as OLIGO (Molecular Biology Insights, Inc., Cascade, Colo.).Important features when designing oligonucleotides to be used asamplification primers include, but are not limited to, an appropriatesize amplification product to facilitate detection (e.g., byelectrophoresis), similar melting temperatures for the members of a pairof primers, and the length of each primer (i.e., the primers need to belong enough to anneal with sequence-specificity and to initiatesynthesis but not so long that fidelity is reduced duringoligonucleotide synthesis). Typically, oligonucleotide primers are 8 to50 nucleotides in length (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).“IS481 primers” and “IS1001 primers” as used herein refers tooligonucleotide primers that specifically anneal to B. pertussis IS481nucleic acid sequences and B. parapertussis IS1001 nucleic acidsequences, respectively, and initiate synthesis therefrom underappropriate conditions.

[0032] Designing oligonucleotides to be used as hybridization probes canbe performed in a manner similar to the design of primers, although themembers of a pair of probes preferably anneal to an amplificationproduct within no more than 5 nucleotides of each other on the samestrand such that FRET can occur (e.g., within no more than 1, 2, 3, or 4nucleotides of each other). This minimal degree of separation typicallybrings the respective fluorescent moieties into sufficient proximitysuch that FRET can occur. It is to be understood, however, that otherseparation distances (e.g., 6 or more nucleotides) are possible providedthe fluorescent moieties are appropriately positioned relative to eachother (for example, with a linker arm) such that FRET can occur. Inaddition, probes can be designed to hybridize to targets that contain amutation or polymorphism, thereby allowing differential detection basedon either absolute hybridization of different pairs of probescorresponding to the particular species to be distinguished ordifferential melting temperatures between, for example, members of apair of probes and each amplification product corresponding to thespecies to be distinguished. As with oligonucleotide primers,oligonucleotide probes usually have similar melting temperatures, andthe length of each probe must be sufficient for sequence-specifichybridization to occur but not so long that fidelity is reduced duringsynthesis. Oligonucleotide probes are 8 to 50 nucleotides in length(e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, or 50 nucleotides in length). “IS481 probes” and “IS1001probes” as used herein refers to oligonucleotide probes thatspecifically anneal to a B. pertussis IS481 amplification product and aB. parapertussis IS1001 amplification product, respectively.

[0033] Constructs of the invention include vectors containing aBordetella nucleic acid e.g., an IS481 or IS1001 nucleic acid molecule,or fragment thereof. Constructs can be used, for example, as controltemplate nucleic acid molecules. Vectors suitable for use in the presentinvention are commercially available and/or produced by recombinant DNAtechnology methods routine in the art. IS481 or IS1001 nucleic acidmolecules can be obtained, for example, by chemical synthesis, directcloning from the respective Bordetella organism, or by PCRamplification. Constructs suitable for use in the methods of theinvention typically include, in addition to IS481 or IS1001 nucleic acidmolecules, sequences encoding a selectable marker (e.g., an antibioticresistance gene) for selecting desired constructs and/or transformants,and an origin of replication. The choice of vector systems usuallydepends upon several factors, including, but not limited to, the choiceof host cells, replication efficiency, selectability, inducibility, andthe ease of recovery.

[0034] Constructs of the invention containing IS481 or IS1001 nucleicacid molecules can be propagated in a host cell. As used herein, theterm host cell is meant to include prokaryotes and eukaryotes such asyeast, plant and animal cells. Prokaryotic hosts may include E. coli,Salmonella tymphimurium, Serratia marcescens and Bacillus subtilis.Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichiapastoris, mammalian cells such as COS cells or Chinese hamster ovary(CHO) cells, insect cells, and plant cells such as Arabidopsis thalianaand Nicotiana tabacum. A construct of the invention can be introducedinto a host cell using any of the techniques commonly known to those ofordinary skill in the art. For example, calcium phosphate precipitation,electroporation, heat shock, lipofection, microinjection, andviral-mediated nucleic acid transfer are common methods for introducingnucleic acids into host cells. In addition, naked DNA can be delivereddirectly to cells (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466).

[0035] Polymerase Chain Reaction (PCR)

[0036] U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188disclose conventional PCR techniques. PCR typically employs twooligonucleotide primers that bind to a selected nucleic acid template(e.g., DNA or RNA). Primers useful in the present invention includeoligonucleotides capable of acting as a point of initiation of nucleicacid synthesis within IS481 or IS1001 nucleic acid sequences. A primercan be purified from a restriction digest by conventional methods, or itcan be produced synthetically. The primer is preferably single-strandedfor maximum efficiency in amplification, but the primer can bedouble-stranded. Double-stranded primers are first denatured, i.e.,treated to separate the strands. One method of denaturingdouble-stranded nucleic acids is by heating.

[0037] The term “thermostable polymerase” refers to a polymerase enzymethat is heat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

[0038] If the B. pertussis or B. parapertussis template nucleic acid isdouble-stranded, it is necessary to separate the two strands before itcan be used as a template in PCR. Strand separation can be accomplishedby any suitable denaturing method including physical, chemical orenzymatic means. One method of separating the nucleic acid strandsinvolves heating the nucleic acid until it is predominately denatured(e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). Theheating conditions necessary for denaturing template nucleic acid willdepend, e.g., on the buffer salt concentration and the length andnucleotide composition of the nucleic acids being denatured, buttypically range from about 90° C. to about 105° C. for a time dependingon features of the reaction such as temperature and the nucleic acidlength. Denaturation is typically performed for about 30 sec to 4 min.

[0039] If the double-stranded nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence on the template nucleicacid. The temperature for annealing is usually from about 35° C. toabout 65° C. Annealing times can be from about 10 secs to about 1 min.The reaction mixture is then adjusted to a temperature at which theactivity of the polymerase is promoted or optimized, i.e., a temperaturesufficient for extension to occur from the annealed primer to generateproducts complementary to the template nucleic acid. The temperatureshould be sufficient to synthesize an extension product from each primerthat is annealed to a nucleic acid template, but should not be so highas to denature an extension product from its complementary template(e.g., the temperature for extension generally ranges from about 40° to80° C.). Extension times can be from about 10 secs to about 5 mins.

[0040] PCR assays can employ template nucleic acid such as DNA or RNA,including messenger RNA (mRNA). The template nucleic acid need not bepurified; it may be a minor fraction of a complex mixture, such as B.pertussis or B. parapertussis nucleic acid contained in human cells. DNAor RNA may be extracted from a biological sample such as nasopharyngealswabs, nasopharyngeal aspirates, and throat swabs by routine techniquessuch as those described in Diagnostic Molecular Microbiology: Principlesand Applications (Persing et al. (eds), 1993, American Society forMicrobiology, Washington D.C.). Template nucleic acids can be obtainedfrom any number of sources, such as plasmids, or natural sourcesincluding bacteria, yeast, viruses, organelles, or higher organisms suchas plants or animals.

[0041] The oligonucleotide primers are combined with PCR reagents underreaction conditions that induce primer extension. For example, chainextension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH8.3), 1.5 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured templateDNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase,and 10% DMSO). The reactions usually contain 150 to 320 μM each of dATP,dCTP, dTTP, dGTP, or one or more analogs thereof.

[0042] The newly synthesized strands form a double-stranded moleculethat can be used in the succeeding steps of the reaction. The steps ofstrand separation, annealing, and elongation can be repeated as often asneeded to produce the desired quantity of amplification productscorresponding to the target nucleic acid molecule. The limiting factorsin the reaction are the amounts of primers, thermostable enzyme, andnucleoside triphosphates present in the reaction. The cycling steps(i.e., denaturation, annealing, and extension) are preferably repeatedat least once. For use in detection, the number of cycling steps willdepend, e.g., on the nature of the sample. If the sample is a complexmixture of nucleic acids, more cycling steps will be required to amplifythe target sequence sufficient for detection. Generally, the cyclingsteps are repeated at least about 20 times, but may be repeated as manyas 40, 60, or even 100 times.

[0043] Fluorescent Resonance Energy Transfer (FRET)

[0044] FRET technology (see, for example, U.S. Pat. Nos. 4,996,143,5,565,322, 5,849,489, and 6,162,603) is based on the concept that when adonor and a corresponding acceptor fluorescent moiety are positionedwithin a certain distance of each other, energy transfer takes placebetween the two fluorescent moieties that can be visualized or otherwisedetected and/or quantitated. Two oligonucleotide probes, each containinga fluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the target nucleic acid sequence. Uponhybridization of the oligonucleotide probe to the amplification productat the appropriate positions, a FRET signal is generated. Hybridizationtemperatures can range from about 35° C. to about 65° C. for about 10seconds to about 1 minute.

[0045] Fluorescent analysis can be carried out using, for example, aphoton counting epifluorescent microscope system (containing theappropriate dichroic mirror and filters for monitoring fluorescentemission at the particular range), a photon counting photomultipliersystem or a fluorometer. Excitation to initiate energy transfer can becarried out with an argon ion laser, a high intensity mercury (Hg) arclamp, a fiber optic light source, or other high intensity light sourceappropriately filtered for excitation in the desired range.

[0046] As used herein with respect to donor and corresponding acceptorfluorescent moieties, “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radiative energytransfer can be produced therebetween.

[0047] Fluorescent donor and corresponding acceptor moieties aregenerally chosen for (a) high efficiency Förster energy transfer; (b) alarge final Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

[0048] Representative donor fluorescent moieties that can be used withvarious acceptor fluorescent moieties in FRET technology includefluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate,Lucifer Yellow VS,4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC™-Red 640, LC™-Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0049] The donor and acceptor fluorescent moieties can be attached tothe appropriate probe oligonucleotide via a linker arm. The length ofthe linker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm for the purpose of the present invention is the distance inAngstroms (Å) from the nucleotide base to the fluorescent moiety. Ingeneral, a linker arm is from about 10 to about 25 Å. The linker arm maybe of the kind described in WO 84/03285. WO 84/03285 also disclosesmethods for attaching linker arms to a particular nucleotide base, andalso for attaching fluorescent moieties to a linker arm.

[0050] An acceptor fluorescent moiety such as an LC™-Red 640-NHS-estercan be combinated with C6-Phosphoramidites (available from ABI (FosterCity, Calif.) or Glen Research (Sterling, Va.)) to produce, for example,LC-Red 640-Phosphoramidite. Frequently used linkers to couple a donorfluorescent moiety such as fluorescein to an oligonucleotide includethiourea linkers (FITC-derived, for example, fluorescein-CPG's from GlenResearch or ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPG's that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

[0051] Detection of B. pertussis and/or B. parapertussis

[0052] The present invention provides methods for detecting the presenceor absence of B. pertussis and/or B. parapertussis in a biologicalsample from an individual. The methods include performing at least onecycling step that first includes contacting the sample with a pair ofIS481 and/or IS1001 primers to produce an IS481 amplification product ifB. pertussis is present in the sample, and/or an IS1001 amplificationproduct if B. parapertussis is present in the sample. Each of the IS481or IS1001 primers anneals to a target within or adjacent to a IS481 orIS1001 nucleic acid molecule, respectively, such that at least a portionof each amplification product contains nucleic acid sequencecorresponding to IS481 or IS1001, respectively. More importantly, theamplification product should contain the nucleic acid sequences that arecomplementary to the IS481 or IS1001 probes, respectively. Each cyclingstep further includes contacting the sample with a pair of IS481 and/orIS1001 probes. According to the invention, one member of each pair ofthe IS481 and IS1001 probes is labeled with a donor fluorescent moietyand the other is labeled with a corresponding acceptor fluorescentmoiety. The presence or absence of FRET between the donor fluorescentmoiety of the first IS481 or IS1001 probe and the corresponding acceptorfluorescent moiety of the second IS481 or IS1001 probe, respectively, isdetected upon hybridization of the probes to the respectiveamplification product. Multiple cycles of amplification andhybridization are performed, preferably in a thermocycler.

[0053] The methods of the invention can be performed individually todetect either B. pertussis or B. parapertussis, but combining theprimers and probes in a single assay to detect the repetitive insertionmolecules (IS481/IS1001) of B. pertussis and B. parapertussis provides arapid and sensitive test that can distinguish between the species in asingle reaction. Representative biological samples that can be used inpracticing the methods of the invention include nasopharyngeal swabs,nasopharyngeal aspirates, throat swabs, or any biological specimen orswab containing ciliated respiratory epithelium that has the potentialto harbor Bordetella species. Biological samples are generally processed(e.g., by nucleic acid extraction methods known in the art) to releaseBordetella nucleic acid.

[0054] As used herein, “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., IS481 or IS1001nucleic acid molecules). Amplifying a nucleic acid molecule typicallyincludes denaturing the template nucleic acid, annealing primers to thetemplate nucleic acid at a temperature that is below the meltingtemperatures of the primers, and enzymatically elongating from theprimers to generate an amplification product. Amplification typicallyrequires the presence of deoxyribonucleoside triphosphates, a DNApolymerase enzyme (e.g., Platinum® Taq) and an appropriate buffer and/orco-factors for optimal activity of the polymerase enzyme (e.g., MgCl₂and/or KCl).

[0055] If amplification of Bordetella nucleic acid occurs and anamplification product is produced, the step of hybridizing results in adetectable signal based upon FRET between the members of the pair ofprobes. As used herein, “hybridizing” refers to the annealing of probesto an amplification product. Hybridization conditions typically includea temperature that is below the melting temperature of the probes butthat avoids non-specific hybridization of the probes.

[0056] Melting curve analysis is an additional step that can be includedin a cycling profile. Melting curve analysis is based on the fact thatDNA melts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the IS481 and IS1001 probes from therespective amplification product(s) can confirm the presence or absenceof B. pertussis and B. parapertussis in the sample, and can distinguishbetween B. pertussis and B. parapertussis. Alternatively, a Lightcycler™apparatus allows for multiple wavelengths to be measured simultaneously.Therefore, the second IS481 and IS1001 probe can be labeled withdifferent acceptor fluorescent moieties (e.g., LC-Red 640 and LC-Red705), thereby providing a method of distinguishing between B. pertussisand B. parapertussis based on differential FRET signals.

[0057] Generally, the presence of FRET indicates the presence of B.pertussis and/or B. parapertussis in the biological sample, and theabsence of FRET indicates the absence of B. pertussis and B.parapertussis in the biological sample. Using the methods disclosedherein, detection of FRET within 40 cycles (e.g., within 30, 25, or 20cycles) is indicative of a B. pertussis and/or B. parapertussisinfection. A positive result indicates the presence of nucleic acid fromB. pertussis and/or B. parapertussis in the biological sample. In somecases, a positive result will be positive for both B. pertussis and B.parapertussis. A negative result indicates the absence of detectable DNAin the specimen submitted for analysis, but does not negate thepossibility of the organism's presence in very small quantities. Anegative result can occur when inhibitory substances are present in thespecimen (studies herein have demonstrated 14% of nasopharyngealspecimens contain unknown PCR-inhibitory components). Inadequatespecimen collection, transportation delays, inappropriate transportationconditions, or use of certain collection swabs (calcium alginate oraluminum shaft) are all conditions that can affect the success and/oraccuracy of the test result.

[0058] Methods of the invention also can be used for vaccine efficacystudies or epidemiology studies of either or both B. pertussis and B.parapertussis. For example, an attenuated B. pertussis or B.parapertussis in a vaccine can be detected using the methods of theinvention during the time when bacteria is still present in anindividual. For such vaccine efficacy studies, the methods of theinvention can be used to determine, for example, the persistence of anattenuated strain of B. pertussis or B. parapertussis used in a vaccine,or can be performed in conjunction with an additional assay such as aserologic assay to monitor an individual's immune response to such avaccine. In addition, methods of the invention can be used todistinguish one B. pertussis or B. parapertussis strain from another forepidemiology studies of, for example, the origin or severity of anoutbreak of B. pertussis or B. parapertussis, respectively.

[0059] Methods of the invention are highly sensitive and highlyspecific. The real-time PCR method disclosed herein is far moresensitive than culture and DFA and superior to the conventional PCR dueto the ability to differentiate between two species of Bordetella. Themethods of the invention do not require gel electrophoresis or Southernhybridization, making the methods described herein much more rapid thanany Bordetella detection method currently available. Rapid diagnosisleading to treatment with antibiotics can prevent potentially seriousconsequences from Bordetella respiratory infections.

[0060] Within each thermocycler run, control samples are cycled as well.Positive control samples can amplify Bordetella nucleic acid controltemplate (other than the IS481 or IS1001 nucleic acid) using, forexample, control primers and control probes. Positive control samplescan also amplify, for example, a plasmid construct containing IS481and/or IS1001. Such a plasmid control can be amplified internally (e.g.,within each sample) or in a separate sample run side-by-side with thepatients' samples. The use of such controls can identify false-negativesdue, for example, to the inhibition of PCR observed with some samples.Each thermocycler run should also include a negative control that, forexample, lacks template DNA.

[0061] In an embodiment, the methods of the invention include steps toavoid contamination. For example, an enzymatic method utilizinguracil-DNA glycosylase is described in U.S. Pat. Nos. 5,035,996,5,683,896 and 5,945,313 to reduce or eliminate contamination between onethermocycler run and the next. In addition, standard laboratorycontainment practices and procedures are desirable when performingmethods of the invention. Containment practices and procedures include,but are not limited to, separate work areas for different steps of amethod, containment hoods, barrier filter pipette tips and dedicated airdisplacement pipettes. Consistent containment practices and proceduresby personnel are necessary for accuracy in a diagnostic laboratoryhandling clinical samples.

[0062] Conventional PCR methods in conjunction with FRET technology canbe used to practice the methods of the invention. In one embodiment, aLightCycler™ instrument is used. A detailed description of theLightCycler™ System and real-time and on-line monitoring of PCR can befound at http://biochem.roche.com/lightcycler. The following patentapplications describe real-time PCR as used in the LightCycler™technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler™instrument is a rapid thermocycler combined with a microvolumefluorometer utilizing high quality optics. This rapid thermocyclingtechnique uses thin glass cuvettes as reaction vessels. Heating andcooling of the reaction chamber are controlled by alternating heated andambient air. Due to the low mass of air and the high ratio of surfacearea to volume of the cuvettes, very rapid temperature exchange ratescan be achieved within the LightCycler™ thermal chamber. Addition ofselected fluorescent dyes to the reaction components allows the PCR tobe monitored in real-time and on-line. Furthermore, the cuvettes serveas an optical element for signal collection (similar to glass fiberoptics), concentrating the signal at the tip of the cuvettes. The effectis efficient illumination and fluorescent monitoring of microvolumesamples.

[0063] The LightCycler™ carousel that houses the cuvettes can be removedfrom the instrument. Therefore, samples can be loaded outside of theinstrument (in a PCR Clean Room, for example). In addition, this featureallows for the sample carousel to be easily cleaned and sterilized. Thefluorimeter, as part of the LightCycler™ apparatus, houses the lightsource. The emitted light is filtered and focused by an epi-illuminationlens onto the top of the cuvettes. Fluorescent light emitted from thesample is then focused by the same lens, passed through a dichroicmirror, filtered appropriately, and focused onto data-collectingphotohybrids. The optical unit currently available in the LightCycler™instrument (Roche Molecular Biochemicals, Catalog No. 2 011 468)includes three band-pass filters (530 nm, 640 nm, and 710 nm), providingthree-color detection and several fluorescence acquisition options. Datacollection options include once per cycling step monitoring, fullycontinuous single-sample acquisition for melting curve analysis,continuous sampling (in which sampling frequency is dependent on samplenumber) and/or stepwise measurement of all samples after definedtemperature interval.

[0064] The LightCycler™ can be operated using a PC workstation and canutilize a Windows NT operating system. Signals from the samples areobtained as the machine positions the cuvettes sequentially over theoptical unit. The software can display the fluorescence signals inreal-time immediately after each measurement. Fluorescent acquisitiontime is 10-100 milliseconds (msec). After each cycling step, aquantitative display of fluorescence vs. cycle number can be continuallyupdated for all samples. The data generated can be stored for furtheranalysis.

[0065] A common FRET technology format utilizes two hybridizationprobes. Each probe can be labeled with a different fluorescent moietyand are generally designed to hybridize in close proximity to each otherin a target DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler™ Instrument. During FRET, thefluorescein transfers its energy to an acceptor fluorescent moiety suchas LightCycler™-Red 640 (LC™-Red 640) or LightCycler™-Red 705 (LC™-Red705). The acceptor fluorescent moiety then emits light of a longerwavelength, which is detected by the optical detection system of theLightCycler™ instrument. Efficient FRET can only take place when thefluorescent moieties are in direct local proximity and when the emissionspectrum of the donor fluorescent moiety overlaps with the absorptionspectrum of the acceptor fluorescent moiety. The intensity of theemitted signal can be correlated with the number of original target DNAmolecules (e.g., the number of B. pertussis or B. parapertussisorganisms).

[0066] Another FRET technology format utilizes TaqMan® technology todetect the presence or absence of an amplification product, and hence,the presence or absence of B. pertussis or B. parapertussis. TaqMan®technology utilizes one single-stranded hybridization probe labeled withtwo fluorescent moieties. When a first fluorescent moiety is excitedwith light of a suitable wavelength, the absorbed energy is transferredto a second fluorescent moiety according to the principles of FRET. Thesecond fluorescent moiety is generally a quencher molecule. During theannealing step of the PCR reaction, the labeled hybridization probebinds to the target DNA (i.e., the amplification product) and isdegraded by the 5′ to 3′ exonuclease activity of the Taq Polymeraseduring the subsequent elongation phase. As a result, the excitedfluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected. By way ofexample, an ABI PRISM® 7700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.) uses TaqMan® technology, and issuitable for performing the methods described herein for detectingBordetella. Information on PCR amplification and detection using an ABIPRISM® 770 system can be found athttp://www.appliedbiosystems.com/products.

[0067] Yet another FRET technology format utilizes molecular beacontechnology to detect the presence or absence of an amplificationproduct, and hence, the presence or absence of Bordetella. Molecularbeacon technology uses a hybridization probe labeled with a donorfluorescent moiety and an acceptor fluorescent moiety. The acceptorfluorescent moiety is generally a quencher, and the fluorescent labelsare typically located at each end of the probe. Molecular beacontechnology uses a probe oligonucleotide having sequences that permitsecondary structure formation (e.g., a hairpin). As a result ofsecondary structure formation within the probe, both fluorescentmoieties are in spatial proximity when the probe is in solution. Afterhybridization to the target nucleic acids (i.e., amplificationproducts), the secondary structure of the probe is disrupted and thefluorescent moieties become separated from one another such that afterexcitation with light of a suitable wavelength, the emission of thefirst fluorescent moiety can be detected.

[0068] As an alternative to detection using FRET technology, anamplification product can be detected using a nucleic acid binding dyesuch as a fluorescent DNA binding dye (e.g., SYBRGreenI® or SYBRGold®(Molecular Probes)). Upon interaction with the double-stranded nucleicacid, such nucleic acid binding dyes emit a fluorescence signal afterexcitation with light at a suitable wavelength. A nucleic acid bindingdye such as a nucleic acid intercalating dye also can be used. Whennucleic acid binding dyes are used, a melting curve analysis is usuallyperformed for confirmation of the presence of the amplification product.

[0069] It is understood that the present invention is not limited by theconfiguration of one or more commercially available instruments.

[0070] Articles of Manufacture

[0071] The invention further provides for articles of manufacture todetect B. pertussis and/or B. parapertussis. An article of manufactureaccording to the present invention can include primers and probes usedto detect B. pertussis or B. parapertussis, together with suitablepackaging materials. Representative primers and probes for detection ofB. pertussis are capable of hybridizing to IS481 nucleic acid molecules.Representative primers and probes for detection of B. parapertussis arecapable of hybridizing to IS1001 nucleic acid molecules. Methods ofdesigning primers and probes are disclosed herein, and representativeexamples of primers and probes that amplify and differentially detect toB. pertussis and B. parapertussis nucleic acid molecules are providedherein.

[0072] Articles of manufacture of the invention can also include one ormore fluorescent moieties for labeling the probes or, alternatively, theprobes supplied with the kit can be labeled. For example, an article ofmanufacture may include a donor fluorescent moiety for labeling one ofthe IS481 or IS1001 probes and a corresponding acceptor fluorescentmoiety for labeling the other IS481 or IS1001 probe, respectively.Examples of suitable FRET donor fluorescent moieties and correspondingacceptor fluorescent moieties are provided herein.

[0073] Articles of manufacture of the invention also can contain apackage insert or package label having instructions thereon for usingthe IS481 primers and probes to detect the presence or absence of B.pertussis in a biological sample and, likewise, using the IS1001 primersand probes to detect the presence or absence of B. parapertussis in asample. Such a package insert may further contain instructions thereonfor using IS481 and IS1001 probes to distinguish between B. pertussisand B. parapertussis within the same biological sample. Articles ofmanufacture may additionally include reagents for carrying out themethods disclosed herein (e.g., buffers, polymerase enzymes, co-factors,or agents to prevent contamination). Such reagents may be specific forone of the commercially available instruments described herein.

[0074] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 DNA Extraction and Bordetella LightCycler™ Assay #1

[0075] Recovery of Bordetella from nasopharyngeal swabs was achieved byswabbing the nasopharynx with a nylon swab having an aluminum shaft andtransported in media. Upon arrival at Mayo, the swab was placed in 500μl Sample Buffer (Reagent A) in a 1.5 ml microcentrifuge tube and storedat 2-8° C. 200 μl of the specimen in Sample Buffer was used for DNAextraction and the remainder was saved at −70° C. for future use. SampleBuffer was added to nasopharyngeal aspirates to bring the volume up to500 μl.

[0076] The DNA sample was taken into a ‘PCR Set-Up Room’ and 200 μl ofthe sample was transferred into 2.0 ml microcentrifuge tubes. DNAextraction was performed in an ‘Extraction PCR Workstation’ using anIsoQuick DNA Extraction Kit (ORCA Research, Inc.; Bothell, Wash.;Catalog #217539). Pellet Paint™ NF Co-Precipitant (Novagen; Madison,Wis.; Catalog #70748-3) was used in all extractions. For diagnostic labsrunning multiple tests on the same biological sample, refer to ‘NucleicAcid Procedures Shared among Molecular Microbiology Tests’ provided withthe IsoQuick DNA Extraction kit.

[0077] Alternatively, DNA was prepared from a sample by boiling andcentrifugation. Briefly, the swab was rinsed with 200 μl of water thatwas collected in a 2 ml screw cap tube. The tube was centrifuged at13,000×g for 1 min and the supernatant was removed. The pellet wasresuspended in 100 μl of RNase-free water and boiled at 100° C. for 10min. The tube was centrifuged at 13,000×g for 1 min and the supernatantcollected.

[0078] In a ‘PCR Clean Room’, the frozen B. pertussis and B.parapertussis LightCycler™ PCR master-mixes were thawed, vortexedbriefly and centrifuged for 1 minute at 20,800×g. If preparedseparately, the B. pertussis and B. parapertussis master mixes werecombined 1:1 in a 1.5 ml Eppendorf tube and mixed. The amount of timethe reagents were left at room temperature was minimized. TheLightCycler™ carousel was loaded with two cuvettes representing positivecontrols, an appropriate number of negative controls, and the remainderwith patient's samples. 15 μl of the combined Bordetella PCR master-mixwas added to each cuvette using a repeat pipettor.

[0079] The cuvettes containing the Bordetella PCR master-mix weretransferred to a ‘Target Loading PCR Workstation’ and 5 μl of the samplesupernatant was carefully removed and added to the 15 μl of BordetellaPCR master-mix in each LightCycler™ cuvette. The cuvettes were capped.The carousel was transported to a ‘LightCycler™ Area’ and wascentrifuged in the LightCycler™ carousel centrifuge. The carousel wasplaced in the LightCycler™ apparatus and the Bordetella LightCycler™program was run. Samples underwent 40 cycles of: denaturation at about95° C. immediately followed by primers annealing to the template nucleicacid for about 20 secs at about 60° C., and elongation of thenewly-synthesized strands at about 72° C. for about 14 secs. During therun, the specimen names were entered and typed into the LightCycler™software sample table. The run was complete in about one hour.

[0080] After completion of the run, the cuvettes were removed from thecarousel with a cuvette extruder or by turning the carousel upside downand gently loosening the cuvettes until they fell into a collectionbucket. The carousel was decontaminated in DNA-OFF (Daigger; VernonHills, Ill.; Cat. #HX12982) for 1 min, rinsed with de-ionized water andair dried before being returned to the ‘PCR Clean Room’.

[0081] Extreme care was taken to avoid all contact of sample or sampleextracts containing Bordetella DNA with any solutions or portion of theLightCycler™ apparatus prior to PCR amplification. False-positivereactions can occur due to cross-contamination fromBordetella-containing samples. For these reasons, the use of at leastthree separate areas for sample preparation and LightCycler™ setup arerecommended: an area for PCR mix preparation (e.g., a ‘PCR Clean Room’),an area for specimen processing and setting-up the PCR reactions (e.g.,an ‘Extraction PCR Workstation’ or a ‘Target Loading PCR Workstation’),and an area dedicated to the actual amplification reactions (e.g., a‘LightCycler™ Area’). Dedicated pipettes and barrier filter pipette tipscan be used with all air displacement pipettes and careful pipetting canminimize any cross-contamination events.

Example 2 Primers and Probes

[0082] Primers (0.2 μM medium scale synthesis) were ordered from theMayo Molecular Biology Core Facility (Rochester, Minn.). Primers weredried down at 60° C. with vacuum (22 psi), and resuspended in 500 μl to1 ml RNase-free water. Primers were adjusted to 50 μM by measuring theA₂₆₀ of a 1/100 dilution (198 μl water+2 μl, DF=100). The concentrationwas estimated by the following formula: [DF×A₂₆₀×100/number ofbases=μM]. The concentration was adjusted to 50 μM by adding water usingthe following formula: [((μM found/50)×μl remaining)−μl remaining=waterto add]. Primers were mixed (1:1) to make a stock solution containing 25μM of each primer and stored at −20° C.

[0083] Probes were obtained from Idaho Technologies(http://www.idahotech.com/itbiochem/index.html). The probes weresuspended in 1×TE buffer supplied with the probes to a finalconcentration of 20 μM.

[0084] The A₂₆₀ and A₄₉₄ of the fluorescein-labeled probe were measured.The extinction coefficient (e₂₆₀) of the fluorescein-labeled probe wascalculated using nearest neighbor values. The LightCycler™ Probe QC, anExcel spreadsheet, was used to calculate the extinction coefficients andratios.

[0085] The dye-oligonucleotide ratio was determined. The ratio should bebetween 0.8 and 1.2, which indicates that there is one dye moleculepresent for every oligonucleotide molecule. Probes were diluted 1/20 in0.5×TE buffer (pH 8.3) to determine this ratio. The extinctioncoefficient of fluorescein is very sensitive to pH. [DyeμM=(A₄₉₄/68,000)]. [Oligo μM=[A₂₆₀−(A₄₉₄×12,000/68,000)]/e₂₆₀×DF×10⁶].

[0086] The A₂₆₀ and A₆₂₂ of the LC-Red 640-labeled oligonucleotide weremeasured and the predicted extinction coefficient (e₂₆₀) was calculatedusing nearest neighbor values. [Dye μM=(A₆₂₂/110,000)]. [OligoμM=[A₂₆₀−(A₆₂₂×31,000/110,000)]/e₂₆₀×DF×10⁶]. TABLE 1 Primers and probesfor detection of B. pertussis Product SEQ Size ID Type (bp) NameSequences (5′→3′) NO: Primer 234 BP IS694 ccagttcctcaaggacgc 1 Primer234 BP IS905 gagttctggtaggtgtgagcgta 2 Probe BP IS Fcaccgctttacccgaccttaccgc 3 ccac Probe BP IS R gaccaatggcaaggccgaacgctt 4catc

[0087] TABLE 2 Primers and probes for detection of B. parapertussisProduct SEQ Size ID Type (bp) Name Sequences (5′→3′) NO: Primer 200 BPPA375 ggcgatatcaacgggtga 5 Primer 200 BPP A556 cagggcaaactcgtccatc 6Probe BPP F gttcttcgaactgggttggcatac 7 Probe BPP Rgtcaagacgctggacaaggctc 8

Example 3 Bordetella LightCycler™ Assay #1

[0088] LightCycler™ PCR master-mixes were prepared in the ‘PCR CleanRoom’. This room was designed with positive airflow and is operated tominimize contamination with nucleic acid from specimens or positivecontrols. Disposable gowns and gloves were worn at all times.

[0089] LightCycler™ PCR mix was prepared according to the followingchart. B. pertussis IS481 PCR mix and B. parapertussis IS1001 PCR mixwere aliquoted into separate 2.0 ml screw-capped microcentrifuge tubesand stored at −70° C. for up to 6 mo. All reagents were thawed, gentlyvortexed and quick spun prior to use (except for Platinum® Taq, whichwas only quick spun). The LightCycler™ PCR mix was prepared as soon asthe reagents were thawed. LightCycler ™ PCR Master Mix - B. pertussisIS481 Number of reactions => 50 Target volume => 5 Stock Mix IngredientStock Conc. Mix Conc. (μl) Water 456.5 MgCl2 50 mM 4 mM 80 10 ×Platinum ® buffer 10 x 1 X 100 Primers 25 μM 0.75 μM 30 Platinum ® Taq 5U/μl 0.025 U/μl 5 dNTP plus 10 mM 0.2 mM 20 BSA 2 % 0.025 % 12.5 HK-UNG10 % 0.2 % 20 BP IS F probe 20 μM 0.2 μM 10.0 BP IS R probe 20 μM 0.3 μM15.0

[0090] LightCycler ™ Hybridization Master Mix - B. parapertussis IS1001Number of reactions => 50 Target volume => 5 Stock Mix Ingredient StockConc. Mix Conc. (μl) Water 447.5 MgCl2 50 mM 4 mM 80 10 × Platinum ®buffer 10 x 1 X 100 Primers 25 μM 0.75 μM 30 DMSO 100 % 1 % 10Platinum ® Taq 5 U/μl 0.025 U/μl 5 dNTP plus 10 mM 0.2 mM 20 BSA 2 %0.025 % 12.5 HK-UNG 10 % 0.2 % 20 BPP F probe 20 μM 0.2 μM 10.0 BPP Rprobe 20 μM 0.3 μM 15.0

[0091] Alternatively, a single master mix can be generated to detecteither or both B. pertussis or B. parapertussis in a biological sample.LightCycler ™ Hybridization Master Mix B. pertussis IS481 and B.parapertussis IS1001 Number of reactions => 50 Target volume => 5 StockMix Ingredient Stock Conc. Mix Conc. (μl) Water 421.5 MgCl₂ 50 mM 4 mM80 10 × Platinum ® buffer 10 x 1 X 100 Primers (B. pertussis) 25 μM 0.5μM 20 Primers (B. parapertussis) 25 μM 0.5 μM 20 Platinum ® Taq 5 U/μl0.03 U/μl 6 dNTP plus 10 mM 0.2 mM 20 BSA 2 % 0.025 % 12.5 HK-UNG 10 %0.2 % 20 BP F probe 20 μM 0.2 μM 10 BP R probe 20 μM 0.3 μM 15 BPP Fprobe 20 μM 0.2 μM 10 BPP R probe 20 μM 0.3 μM 15

Example 4 Quality Control

[0092] A positive control of both B. pertussis (ATCC #9797) and B.parapertussis (ATCC#15311) were extracted and processed through theLightCycler™ detection in each clinical run. A melting curve analysiswas used to differentiate the two organisms. If amplification of thepositive control was not detected within 4 cycles of the expected numberof cycles for detection of positive controls, or does not amplify, therun was repeated.

[0093] A fresh culture of the ATCC strains of B. pertussis and B.parapertussis were grown on charcoal agar at 37° C. in a CO₂ incubator.Several colonies were resuspended in sterile saline and adjusted to aMacFarland standard of 0.5 (ca.1.5×10⁸ organisms/ml) using the VitekColorimeter (85% T±2). A 10-fold dilution series was prepared usingmolecular grade water (50 μl dilution into 450 μl water). Recoverystudies were performed by adding 20 μl of each dilution of the series toSample Buffer and extracting the DNA. The optimal concentration of B.pertussis and B. parapertussis was determined and a stock solution ofthe appropriately diluted culture was made in molecular grade water andstored at 4° C.

[0094] A positive control was generated by cloning IS481 or IS1001nucleic acid molecules into a vector using the Invitrogen TOPO TACloning kit (Cat. #K4500-01). The 234 bp PCR amplicon of B. pertussisand the 200 bp PCR amplicon of B. parapertussis were each inserted intoa plasmid vector (pCR 2.1-TOPO). The recombinant vector was transformedinto chemically competent E. coli and grown overnight on a LB agar platecontaining 50 μg/ml of kanamycin. The white colonies containing theconfirmed recombinant plasmid were grown overnight in LB brothcontaining kanamycin and purified with the Promega Wizard Plus MiniPrepDNA purification system (Cat. #A7500). The stock concentration of thepositive plasmid control was determined in molecules/μl. A ten-foldserial dilution was prepared using 20 μl of the suspension and 180 μl ofsterile RNAse-free water. This dilution series was carried through untilno amplification product was detected. Each dilution was tested with theBordetella LightCycler™ assay and the optimal positive control dilutionwas determined. A working solution of 1.0 ml of this dilution wasprepared and stored at 4° C.

[0095] Alternatively, the positive control was extracted from a culture(20 μl control plus 180 μl IsoQuick Sample Buffer) and processed inparallel with the clinical specimens to provide a consistent means ofmonitoring assay performance. Negative controls were included in eachclinical run. Negative controls consisted of 5-10% of the batch and wereinterspersed in the LightCycler™ apparatus with patient samples. Thesecontrols tested for hybridization mix contamination andspecimen-to-specimen carryover contamination. If a negative control(s)yielded a positive reaction, extraction reagents were replaced and thesamples and controls from the run in question were re-extracted.

[0096] IsoQuick solution Sample Buffer A was extracted and used as anegative control. This was to confirm that extraction reagents were notcontaminated with previously amplified product. Alternatively, 5 μl ofwater was added directly to the Master Mix and amplified as a negativecontrol. All specimens (patient's and controls) were handled usingUniversal Precautions. Sterile gloves were worn when handling samplesand performing all procedures. Gloves were changed frequently.

[0097] dUTP incorporation and uracil N-glycosylase treatment with athermolabile UNG (Epicentre Technologies; Madison, Wis.; Catalog#HU5910K) were used to prevent amplicon carryover in the LightCycler™assays described herein. Although not required, the routineimplementation of these precautions diminishes the risk offalse-positive results. False-positive results have been a significantand often cited problem in many laboratories using PCR techniques andcan seriously compromise the reliability of testing performed in aclinical environment.

Example 5 Interpreting and Reporting Results

[0098] A clinical specimen that displayed a melting temperature of75°±2° C. was interpreted as positive for B. pertussis and/or a meltingtemperature of 64°±2° C. was interpreted as positive for B.parapertussis DNA.

[0099] The B. pertussis IS481 assay is specific for B. pertussis and apositive signal is reported as B. pertussis. Although the primers andprobes are specific for the insertion sequence of B. pertussis IS481,cross-reactivity with B. holmesii can occur with B. pertussis IS481 PCRassays. Cephalexin, the antibiotic widely used in culture media, has aninhibitory effect on B. holmesiii. In one evaluation, B. holmesiipositivity rate in nasopharyngeal specimens was 0.29%. The clinicalsignificance of B. holmesii has yet to be determined although it hasbeen associated with septicemia, respiratory failure and symptomssimilar to B. pertussis infection (i.e., cough).

[0100] The B. parapertussis IS1001 assay is specific for B.parapertussis species and a positive signal is reported as B.parapertussis. Because the primers and probes are specific for B.parapertussis, and no cross-reactions have been observed with thesereagents, a positive test will provide results of the specific nucleicacid. Therefore, positive results can be reported as B. parapertussis.

[0101] A clinical specimen or control with no melting curve abovebaseline should be interpreted as negative for the presence of B.pertussis or B. parapertussis DNA. Results are strictly qualitative. Anegative result does not negate the presence of the organism or activedisease. Test results should be used as an aid in diagnosis and not beconsidered a stand-alone diagnostic test. A single assay should not beused as the only criteria to form a clinical conclusion, but resultsshould be correlated with serologic tests, patient symptoms, andclinical presentation.

Example 6 Method Validation

[0102] The LightCycler™ PCR assay for detection of B. pertussis and/orB. parapertussis was compared to culture/DFA, to conventional PCR of theIS481 gene of B. pertussis, and to a LightCycler™ PCR assay fordetection of the pertussis toxin gene (PTG). A combined gold standardwas used to compare the LightCycler™ PCR assay to the other detectionmethods. This gold standard is defined as ≧1 positive result in anycombination of results from culture/DFA, PTG, and conventional PCR.

[0103] Compared to culture/DFA and a LightCycler™ PCR assay fordetection of PTG, the LightCycler™ PCR assay for detection of B.pertussis and B. parapertussis using IS481 and IS1001, respectively, was100% sensitive and 72% specific (p<0.0001 using a kappa statistic). Thepositive predictive value (ppv) and the negative predictive value (npv)were 30% and 100%, respectively. Compared to the LightCycler™ PCR assay,conventional PCR of the IS481 gene had a sensitivity of 96%, specificityof 82%, ppv of 83%, and an npv of 95% (p=0.0654). Compared toLightCycler™ PCR and PTG LightCycler™ PCR, culture/DFA had a sensitivityof 25%, specificity of 100%, ppv of 100%, and an npv of 70% (p<0.0001).Compared to LightCycler™ PCR and culture/DFA, the PTG LightCycler™ assayhad a sensitivity of 28%, specificity of 100%, ppv of 100%, and an npvof 71% (p<0.0001). Using dilutions of well-characterized American TypeCulture Collection (ATCC) and CDC positive controls, the sensitivity ofthe IS481/IS1001 assay was 1 organism/μl for both the detection of B.pertussis and B. parapertussis.

[0104] A low level positive control of B. pertussis and B. parapertussiswas run multiple times within a run, two times within a day and on threeconsecutive days. The variability was determined to be in the acceptablerange of ±4 cycles. The analytical detection limit of the LightCycler™PCR assay, using dilutions of a McFarland 0.5 standard of freshcultures, was 1 organism per reaction. The average number of templatesper organism was 80 in B. pertussis and 20 in B. parapertussis.

[0105] Ninety-two IsoQuick extracted nasopharyngeal samples were spikedwith B. pertussis and B. parapertussis and tested for the presence ofinhibitors. 13 samples did not amplify under such conditions, giving aninhibition rate of 14%. The choice of transport swab and medium mayaffect inhibition (e.g., calcium alginate swabs, cotton swabs, andaluminum shaft swabs may be inhibitory to PCR).

Example 7 Bordetella LightCycler Assay #2 with Recovery Template

[0106] 200 μl sterile water was added to an original tube containing anasopharyngeal swab, and the tube was vortexed well. 200 μl of the swabmaterial was transferred to a screw-capped tube containing 4 μl ofrecovery template (5×10³ targets/μl), and the tube was capped and mixedbriefly. Recovery template is modified template nucleic acid that isco-amplified in the same tube by the same set of primers used to amplifytemplate nucleic acid. Recovery template, however, uses different probesfor detection. The probes that hybridize to the recovery template(apoE-F and apoE 705) are labeled with fluorescein and LC-Red 705 sothat amplification of the recovery template is measured on a differentchannel than the channel used to measure amplification of the template.Recovery template can be used to identify samples containing aninhibitory component.

[0107] 100 μl of STAR Buffer (Roche Molecular Diagnostics, Indianapolis,Ind.) was placed into a MagNA Pure sample cartridge. 100 μl of the swabsample (containing recovery template at a final concentration of 1×10²targets/μl) was transferred into extraction wells, and the DNA extractedusing MagNA Pure and the LightCycler Total Nucleic Acid Isolation kit.

[0108] The remaining 100 μl of the swab sample was placed in a 95° C.(±5° C.) heat block for 10 min. The tube was centrifuged for 3 min at20,000×g to pellet any particulate material.

Example 8 Control Samples

[0109] Several types of positive controls were used. For a positivecontrol of each organism, B. pertussis (ATCC Accession No. 9797) or B.parapertussis (ATCC Accession No. 15311) were grown on charcoal agar anddiluted to a McFarland 0.5 (1.5×10⁸ cells/ml). The cultures were furtherdiluted to a working concentration of 1.5×10⁶ cells/ml and used ascontrols for extraction and amplification reactions of template from theboiled lysate procedure and from the MagNA Pure extraction. A positivecontrol corresponding to each Bordetella organism included 198 μlsterile water+2 μl organism control (1.5×10⁶ cells/μi)+4 μl recoverytemplate (5×10³ targets/μl).

[0110] In addition to the organism controls, a plasmid containingBordetella sequences was used as a positive control. The plasmidcontains sequences from both IS481 (B. pertussis) and IS1001 (B.parapertussis) (Roche Molecular Diagnostics). A positive extractioncontrol corresponding to the plasmid included 198 μl sterile water+2 μlplasmid (2×10³ targets/μl)+4 μl recovery template (5×10³ targets/μl).

[0111] A negative control included 200 μl sterile water+4 μl recoverytemplate (5×10³ targets/μl).

Example 9 Bordetella LightCycler Assay #2

[0112] Tables 3 and 4 describe the sequences of the primers and probesused for detection of B. pertussis and B. parapertussis, respectively.TABLE 3 SEQ ID Type Name Sequences (5′→3′) NO: Primer BP IS1ccagttcctcaaggacgc  1 Primer BP IS2 gagttctggtaggtgtgagcgta  2 ProbeBP-F(WT) caccgctttacccgaccttaccg  3 cccac Probe BP853mm29gaccaatggcaaggctcgaacgc 11 ttcatc

[0113] TABLE 4 Type Name Sequences (5′→3′) SEQ ID NO: Primer BPP IS1ggcgatatcaacgggtga  5 Primer BPP IS2 cagggcaaactcgtccatc  6 Probe VPP03ggttggcataccgtcaaga 12 Probe VPP04 gctggacaaggctcg 13

[0114] The components of the Bordetella LightCycler Assay #2 reactionmix are as follows. Reagent Volume Water 11 μl Primer/Probe Mix^(a)  2μl FastStart DNA Master Hybridization Probes^(b)  2 μl Total 15 μl

[0115] As an alternative to adding the recovery template to the sampleprior to extraction, recovery template can be added to the PCR reactionmix at a final concentration of 5×10³ targets/μl.

[0116] 5 μl from the boiled lysate, the MagNA Pure extraction, or thepositive or negative control samples, was mixed with 15 μl of theBordetella LightCycler PCR reaction mix described above, placed in aLightCycler carousel, and amplified as follows. A. Initial 95° C. 10 min45 cycles B. PCR 95° C. 10 sec 55° C. 15 sec Single signal 72° C. 15 secC. Melt 95° C.  0 sec  20°/sec 59° C. 20 sec  20°/sec 45° C. 20 sec0.2°/sec 85° C.  0 sec 0.1°/sec Continuous Signal D. Melt 2 95° C.  0sec  20°/sec 59° C. 20 sec  20°/sec 45° C. 20 sec 0.2°/sec 85° C.  0 sec0.2°/sec Continuous Signal E. Cool 40° C. 10 sec

[0117] Results of the experiments are shown in Table 5. The addition ofthe recovery template to the specimens prior to nucleic acid extractiondid not inhibit the extraction or the amplification of a product. Asample was considered to contain an inhibitory component if the recoverytemplate amplification product was not generated in the absence oftarget template. TABLE 5 Bordetella LightCycler Assay #2 BordetellaMagNA Pure Boil (w/ LightCycler (w/recovery recovery Sample Assay #1MagNA Pure template) Boil template) 1 − − + − + 2 + B. pert − B. pert −3 + B. pert + B. pert + 4 − − + − + 5 − − + − + 6 − − + − + 7 + B.para + B. para + 8 + B. pert + B. pert − 9 + B. pert + B. pert − 10 −− + − + 11 − − + − + 12 + B. pert + B. pert + 13 − − + − + 14 + B.pert + B. pert + 15 − − + − + 16 − − + − + 17 − − + − + 18 + B. pert +B. pert + 19 − − + − + 20 + B. pert + B. pert + 21 − − + − + 22 + B.pert − B. pert − 23 + B. pert + B. pert + 24 + B. pert + B. pert + 25 −− + − + 26 − − + − + 27 − − + − + 28 − − + − + 29 + B. para + B. para +30 + B. pert + B. pert +

Example 10 Specificity of Bordetella LightCycler Assay #2

[0118] Experiments were performed to determine if the Bordetella primersand probes cross-reacted with nucleic from similar organisms or fromorganisms commonly found in the specimens tested.

[0119] 5 μl from the boiled lysate or from the MagNA Pure extraction wasadded to 15 μl of the PCR reaction mix described above in Example 9.Samples were placed into a LightCycler carousel and cycled as describedabove in Example 9. Nucleic acid had previously been shown to beamplifiable in the bacterial organisms listed in Table 6 using 16S rRNAamplification by conventional or LightCycler PCR assays. 2×10³targets/μl of the positive control plasmid described above in Example 8was used. TABLE 6 Organism ResultHZ,1/32 Pseudomonas aeruginosa −Chlamydia pneumoniae − Klebsiella pneumoniae − Escherichia coli −Haemophilus influenza − Aeromonas species − Staphylococcus aureus −Legionella Jordanis − S. maltophilia − Klebsiella oxytoca − Pseudomonascepacia − Staphylococcus epidermidis − Neisseria gonorrhoeae −Pseudomonas fluorescens − C. pseudodiptheriae − Morganella species −Proteus vulgaris − Mycoplasma pneumonia − Campylobacter jejuni − M.catarrhalis − Human DNA − Bordetella pertussis + Legionella pneumophila− Bordetella bronchioseptica − Neisseria meningitidis − Bordetellaholmesii + Acinetobacter species − Proteus mirabilis − Corynebacteriumdiphtheriae − Bordetella parapertussis +

[0120] Other than the B. pertussis and B. parapertussis positivecontrols and the closely related B. holmesii, which was previouslydetected with the Bordetella Lightcycler Assay #1, none of the organismsshown in Table 6 cross-reacted with the Bordetella primers and probesused in the Bordetella LightCycler Assay #2.

Example 11 Diagnositic Sensitivity of the Bordetella LightCycler Assay#2

[0121] Experiments were performed to determine the sensitivity andspecificity of the Bordetella LightCycler Assay #2 compared to cultureand other amplification methods. 110 nasopharyngeal swabs from patientsamples sent in for Bordetella testing were examined using a culturemethod, and a portion of those samples were analyzed by a conventionalPCR method and the Bordetella LightCycler Assays #1 and #2 disclosedherein. The nasopharyngeal swabs were initially cultured on charcoalagar plates containing cephalexin and blood agar, incubated at 37° C.and examined daily for 5 days for the presence of B. pertussis and/or B.parapertussis.

[0122] After swiping on agar plates, the nasopharyngeal swabs wereswished in a tube containing 500 μl sterile water. 200 μl was removedfrom the tube (n=35) and processed by boiling at 100° C. for 10 min.After boiling, the sample was centrifuged for 1 minute at 20,800×g. TheDNA present in 200 μl of the remaining nasopharyngeal swab sample wasextracted using the IsoQuick Nucleic Acid Extraction Kit (ORCA Research,Inc., Bothell, Wash.). These samples were stored at −20° C. for up to 48months.

[0123] Using 99 of the 110 samples described above, 5 μl of the boiledlysate or the IsoQuick extraction eluate was added to 15 μl of the PCRReaction Mix described above in Example 3. The samples were analyzed bythe Bordetella LightCycler Assay described above in Example 1(Bordetella LightCycler Assay #1).

[0124] Another 5 μl of the boiled lysate or the IsoQick extractioneluate was added to 15 μl of the PCR Reaction Mix described above inExample 8. The samples were analyzed by the Bordetella LightCycler Assaydescribed above in Example 8 (Bordetella LightCycler Assay #2).

[0125] A conventional PCR assay was used and amplified IS481 nucleicacid sequences. Following amplification by PCR, the sample waselectrophoresed on an agarose gel, Southern blotted, and detected byenzyme chemiluminescence (Amersham Corporation, Arlington Heights,Ill.). The conventional PCR protocol for detecting B. pertussis hadapproximately a 2-5 day turnaround time.

[0126] Results of culture methods versus the Bordetella LightCyclerAssay #2 are shown in Table 7. Results of experiments comparingBordetella LightCycler Assay #1, Bordetella LightCycler Assay #2, andthe conventional PCR assay are shown in Table 8. TABLE 7 Culture B. B.pertussis parapertussis Negative Total Bordetella B. pertussis 18 0 2341 LightCycler B. parapertussis 0 4 1  5 Assay #2 Negative 0 0 59 59Total 18 4 83 105*

[0127] TABLE 8 Bordetella LightCycler Assay #1 B. B. pertussisparapertussis Negative Total Bordetella B. pertussis 38 0 3 41LightCycler B. parapertussis 0 2 1  3 Assay #2 Negative 9 2 44 55 TOTAL47 4 48 99

[0128] Five samples were found to inhibit PCR amplification using theBordetella Lightcycler Assay #2 based upon a lack of detection of therecovery template. The correlation between the Bordetella LightCyclerAssay #1 and the Bordetella LightCycler Assay #2 was good. The integrityof some of the samples was questionable, with nucleic acid samplesarchived for a longer period of time sometimes resulting in a negativeresult.

OTHER EMBODIMENTS

[0129] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 8 1 18 DNA Artificial Sequence Oligonucleotides 1 ccagttcctc aaggacgc18 2 23 DNA Artificial Sequence Oligonucleotides 2 gagttctggt aggtgtgagcgta 23 3 28 DNA Artificial Sequence Oligonucleotides 3 caccgctttacccgacctta ccgcccac 28 4 28 DNA Artificial Sequence Oligonucleotides 4gaccaatggc aaggccgaac gcttcatc 28 5 18 DNA Artificial SequenceOligonucleotides 5 ggcgatatca acgggtga 18 6 19 DNA Artificial SequenceOligonucleotides 6 cagggcaaac tcgtccatc 19 7 24 DNA Artificial SequenceOligonucleotides 7 gttcttcgaa ctgggttggc atac 24 8 22 DNA ArtificialSequence Oligonucleotides 8 gtcaagacgc tggacaaggc tc 22

What is claimed is:
 1. A method for detecting the presence or absence ofBordetella pertussis in a biological sample from an individual, saidmethod comprising: performing at least one cycling step, wherein acycling step comprises an amplifying step and a hybridizing step,wherein said amplifying step comprises contacting said sample with apair of IS481 primers to produce an IS481 amplification product if a B.pertussis IS481 nucleic acid molecule is present in said sample, whereinsaid hybridizing step comprises contacting said sample with a pair ofIS481 probes, wherein the members of said pair of IS481 probes hybridizewithin no more than five nucleotides of each other, wherein a firstIS481 probe of said pair of IS481 probes is labeled with a donorfluorescent moiety and a second IS481 probe of said pair of IS481 probesis labeled with a corresponding acceptor fluorescent moiety; anddetecting the presence or absence of fluorescence resonance energytransfer (FRET) between said donor fluorescent moiety of said firstIS481 probe and said corresponding acceptor fluorescent moiety of saidsecond IS481 probe, wherein the presence of FRET is indicative of thepresence of B. pertussis in said biological sample, and wherein theabsence of FRET is indicative of the absence of B. pertussis in saidbiological sample.
 2. The method of claim 1, wherein said pair of IS481primers comprises a first IS481 primer and a second IS481 primer,wherein said first IS481 primer comprises the sequence 5′-CCA GTT CCTCAA GGA CGC-3′ (SEQ ID NO: 1), and wherein said second IS481 primercomprises the sequence 5′-GAG TTC TGG TAG GTG TGA GCG TA-3′ (SEQ IDNO:2).
 3. The method of claim 1, wherein said first IS481 probecomprises the sequence 5′-CAC CGC TTT ACC CGA CCT TAC CGC CCA C-3′ (SEQID NO:3), and wherein said second IS481 probe comprises the sequence5′-GAC CAA TGG CAA GGC TCG AAC GCT TCA TC-3′ (SEQ ID NO:11).
 4. Themethod of claim 1, wherein the members of said pair of IS481 probeshybridize within no more than two nucleotides of each other.
 5. Themethod of claim 1, wherein the members of said pair of IS481 probeshybridize within no more than one nucleotide of each other.
 6. Themethod of claim 1, wherein said donor fluorescent moiety is fluorescein.7. The method of claim 1, wherein said acceptor fluorescent moiety isselected from the group consisting of LC™-Red 640, LC™-Red 705, Cy5, andCy5.5.
 8. The method of claim 1, wherein said detecting step comprisesexciting said biological sample at a wavelength absorbed by said donorfluorescent moiety and visualizing and/or measuring the wavelengthemitted by said acceptor fluorescent moiety.
 9. The method of claim 1,wherein said detecting comprises quantitating said FRET.
 10. The methodof claim 1, wherein said detecting step is performed after each cyclingstep.
 11. The method of claim 1, wherein said detecting step isperformed in real-time.
 12. The method of claim 1, wherein the presenceof said FRET within 40 cycling steps is indicative of the presence of aB. pertussis infection in said individual.
 13. The method of claim 1,wherein the presence of said FRET within 30 cycling steps is indicativeof the presence of a B. pertussis infection in said individual.
 14. Themethod of claim 1, wherein the presence of said FRET within 25 cyclingsteps is indicative of the presence of a B. pertussis infection in saidindividual.
 15. The method of claim 1, further comprising preventingamplification of a contaminant nucleic acid.
 16. The method of claim 15,wherein said preventing comprises performing said amplifying step in thepresence of uracil.
 17. The method of claim 16, wherein said preventingfurther comprises treating said biological sample with uracil-DNAglycosylase prior to a first amplifying step.
 18. The method of claim 1,wherein said biological sample is selected from the group consisting ofnasopharyngeal swabs, nasopharyngeal aspirates, and throat swabs. 19.The method of claim 1, wherein said cycling step is performed on acontrol sample.
 20. The method of claim 19, wherein said control samplecomprises said portion of said IS481 nucleic acid molecule.
 21. Themethod of claim 1, wherein said cycling step uses a pair of controlprimers and a pair of control probes, wherein said control primers andsaid control probes are other than said IS481 primers and IS481 probes,respectively, wherein a control amplification product is produced ifcontrol template is present in said sample, wherein said control probeshybridize to said control amplification product.
 22. A method fordetecting the presence or absence of Bordetella parapertussis in abiological sample from an individual, said method comprising: performingat least one cycling step, wherein a cycling step comprises anamplifying step and a hybridizing step, wherein said amplifying stepcomprises contacting said sample with a pair of IS1001 primer to producean IS1001 amplification product if a B. parapertussis IS1001 nucleicacid molecule is present in said sample, wherein said hybridizing stepcomprises contacting said biological sample with a pair of IS1001probes, wherein the members of said pair of IS1001 probes hybridizewithin no more than five nucleotides of each other, wherein a firstIS1001 probe of said pair of IS1001 probes is labeled with a donorfluorescent moiety and a second IS1001 probe of said pair of IS1001probes is labeled with a corresponding acceptor fluorescent moiety; anddetecting the presence or absence of FRET between said donor fluorescentmoiety of said first IS1001 probe and said corresponding acceptorfluorescent moiety of said second IS1001 probe, wherein the presence ofFRET is indicative of the presence of B. parapertussis in saidbiological sample, and wherein the absence of FRET is indicative of theabsence of B. parapertussis in said biological sample.
 23. The method ofclaim 22, wherein said pair of IS1001 primers comprises a first IS1001primer and a second IS1001 primer, wherein said first IS1001 primercomprises the sequence 5′-GGC GAT ATC AAC GGG TGA-3′ (SEQ ID NO:5), andwherein said second IS1001 primer comprises the sequence 5′-CAG GGC AAACTC GTC CAT C-3′ (SEQ ID NO:6).
 24. The method of claim 22, wherein saidfirst IS1001 probe comprises the sequence 5′-GGT TGG CAT ACC GTC AAGA-3′ (SEQ ID NO:12), and wherein said second IS1001 probe comprises thesequence 5′-GCT GGA CAA GGC TCG-3′ (SEQ ID NO:13).
 25. A method fordetecting the presence or absence of Bordetella pertussis and/or B.parapertussis in a biological sample from an individual, said methodcomprising: performing at least one cycling step, wherein a cycling stepcomprises an amplifying step and a hybridizing step, wherein saidamplifying step comprises contacting said sample with a pair of IS481primers and a pair of IS1001 primers to produce an IS481 amplificationproduct if a B. pertussis IS481 nucleic acid molecule is present in saidsample and an IS1001 amplification product if a B. parapertussis IS1001nucleic acid molecule is present in said sample, wherein saidhybridizing step comprises contacting said sample with a pair of IS481probes and a pair of IS1001 probes, wherein the members of said pair ofIS481 probes hybridize within no more than five nucleotides of eachother and wherein the members of said pair of IS1001 probes hybridizewithin no more than five nucleotides of each other, wherein a firstIS481 probe of said pair of IS481 probes is labeled with a donorfluorescent moiety and wherein a second IS481 probe of said pair ofIS481 probes is labeled with a corresponding acceptor fluorescentmoiety, wherein a first IS1001 probe of said pair of IS1001 probes islabeled with a donor fluorescent moiety and wherein a second IS1001probe of said pair of IS1001 probes is labeled with a correspondingacceptor fluorescent moiety; and detecting the presence or absence ofFRET between said donor fluorescent moiety of said first IS481 probe andsaid corresponding acceptor fluorescent moiety of said second IS481probe and/or between donor fluorescent moiety of said first IS1001 probeand said corresponding acceptor fluorescent moiety of said second IS1001probe, wherein the presence of FRET is indicative of the presence of B.pertussis and/or B. parapertussis in said biological sample, and whereinthe absence of FRET is indicative of the absence of B. pertussis or B.parapertussis in said biological sample.
 26. The method of claim 25,further comprising: determining the melting temperature between one orboth of said IS481 probes and said IS481 amplification product andbetween one or both of said IS1001 probes and said IS1001 amplificationproduct, wherein said melting temperature(s) confirms said presence orabsence of B. pertussis in said sample and said presence or absence ofB. parapertussis in said sample.
 27. The method of claim 26, whereinsaid melting temperature(s) distinguish between B. pertussis and B.parapertussis in said sample.
 28. The method of claim 25, wherein saidacceptor fluorescent moiety of said second IS481 probe and said acceptorfluorescent moiety of said second IS1001 probe are different.
 29. Anarticle of manufacture, comprising: a pair of IS481 primers; a pair ofIS481 probes; and a first donor fluorescent moiety and a correspondingfirst acceptor fluorescent moiety.
 30. The article of manufacture ofclaim 29, wherein said pair of IS481 primers comprise a first IS481primer and a second IS481 primer, wherein said first IS481 primercomprises the sequence 5′-CCA GTT CCT CAA GGA CGC-3′ (SEQ ID NO: 1), andwherein said second IS481 primer comprises the sequence 5′-GAG TTC TGGTAG GTG TGA GCG TA-3′ (SEQ ID NO:2).
 31. The article of manufacture ofclaim 29, wherein said pair of IS481 probes comprises a first IS481probe and a second IS481 probe, wherein said first IS481 probe comprisesthe sequence 5′-CAC CGC TTT ACC CGA CCT TAC CGC CCA C-3′ (SEQ ID NO:3),and wherein said second IS481 probe comprises the sequence 5′-GAC CAATGG CAA GGC TCG AAC GCT TCA TC-3′ (SEQ ID NO:11).
 32. The article ofmanufacture of claim 29, wherein said first IS481 probe is labeled withsaid first donor fluorescent moiety and wherein said second IS481 probeis labeled with said corresponding first acceptor fluorescent moiety.33. The article of manufacture of claim 29, further comprising: a pairof IS1001 primers; a pair of IS1001 probes; and a second donorfluorescent moiety and a corresponding second acceptor fluorescentmoiety.
 34. The article of manufacture of claim 33, wherein said pair ofIS1001 primers comprise a first IS1001 primer and a second IS1001primer, wherein said first IS1001 primer comprises the sequence 5′-GGCGAT ATC AAC GGG TGA-3′ (SEQ ID NO:5), and wherein said second IS1001primer comprises the sequence 5′-CAG GGC AAA CTC GTC CAT C-3′ (SEQ IDNO:6).
 35. The article of manufacture of claim 33, wherein said pair ofIS1001 probes comprise a first IS1001 probe and a second IS1001 probe,wherein said first IS1001 probe comprises the sequence 5′-GGT TGG CATACC GTC AAG A-3′ (SEQ ID NO:12), and wherein said second IS1001 probecomprises the sequence 5′-GCT GGA CAA GGC TCG-3′ (SEQ ID NO:13).
 36. Thearticle of manufacture of claim 33, wherein said pair of S1001 probesare labeled with said second donor fluorescent moiety and saidcorresponding second acceptor fluorescent moiety.
 37. The article ofmanufacture of claim 33, further comprising a package insert havinginstructions thereon for using said pair of IS481 primers, said pair ofIS481 probes, said pair of IS1001 primers and said pair of IS1001 probesto detect the presence or absence of B. pertussis and/or B.parapertussis in a biological sample.
 38. The article of manufacture ofclaim 37, further comprising a package insert having instructionsthereon for using said pair of IS481 probes and said pair of IS1001probes to distinguish between B. pertussis and B. parapertussis in saidbiological sample.
 39. An article of manufacture, comprising: a pair ofIS1001 primers; a pair of IS1001 probes; and a donor fluorescent moietyand a corresponding acceptor fluorescent moiety.
 40. A method fordetecting the presence or absence of B. pertussis in a biological samplefrom an individual, said method comprising: performing at least onecycling step, wherein a cycling step comprises an amplifying step and ahybridizing step, wherein said amplifying step comprises contacting saidsample with a pair of IS481 primers to produce an IS481 amplificationproduct if a B. pertussis IS481 nucleic acid molecule is present in saidsample, wherein said hybridizing step comprises contacting said samplewith an IS481 probe, wherein the IS481 probe is labeled with a donorfluorescent moiety and a corresponding acceptor fluorescent moiety; anddetecting the presence or absence of fluorescence resonance energytransfer (FRET) between said donor fluorescent moiety and said acceptorfluorescent moiety of said IS481 probe, wherein the presence or absenceof FRET is indicative of the presence or absence of B. pertussis in saidsample.
 41. The method of claim 40, wherein said amplification employs apolymerase enzyme having 5′ to 3′ exonuclease activity.
 42. The methodof claim 41, wherein said donor and acceptor fluorescent moieties arewithin no more than 5 nucleotides of each other on said probe.
 43. Themethod of claim 42, wherein said acceptor fluorescent moiety is aquencher.
 44. The method of claim 40, wherein said IS481 probe comprisesa nucleic acid sequence that permits secondary structure formation,wherein said secondary structure formation results in spatial proximitybetween said donor and said acceptor fluorescent moiety.
 45. The methodof claim 44, wherein said acceptor fluorescent moiety is a quencher. 46.A method for detecting the presence or absence of B. pertussis in abiological sample from an individual, said method comprising: performingat least one cycling step, wherein a cycling step comprises anamplifying step and a dye-binding step, wherein said amplifying stepcomprises contacting said sample with a pair of IS481 primers to producean IS481 amplification product if a B. pertussis IS481 nucleic acidmolecule is present in said sample, wherein said dye-binding stepcomprises contacting said IS481 amplification product with a nucleicacid binding dye; and detecting the presence or absence of binding ofsaid nucleic acid binding dye to said amplification product, wherein thepresence of binding is indicative of the presence of B. pertussis insaid sample, and wherein the absence of binding is indicative of theabsence of B. pertussis in said sample.
 47. The method of claim 46,wherein said nucleic acid binding dye is selected from the groupconsisting of SYBRGreenI®, SYBRGold®, and ethidium bromide.
 48. Themethod of claim 47, further comprising determining the meltingtemperature between said IS481 amplification product and said nucleicacid binding dye, wherein said melting temperature confirms saidpresence or absence of said B. pertussis.